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EP3391877A1 - Préparation de nanoparticules lipidiques - Google Patents

Préparation de nanoparticules lipidiques Download PDF

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Publication number
EP3391877A1
EP3391877A1 EP18174687.6A EP18174687A EP3391877A1 EP 3391877 A1 EP3391877 A1 EP 3391877A1 EP 18174687 A EP18174687 A EP 18174687A EP 3391877 A1 EP3391877 A1 EP 3391877A1
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Prior art keywords
lipid
mixing
buffer
solution
organic
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German (de)
English (en)
Inventor
Varun Kumar
Robert K. Prud'homme
Paul A. Burke
Marian E. Gindy
David J. Mathre
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Princeton University
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Princeton University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1277—Preparation processes; Proliposomes

Definitions

  • Liposomes are typically prepared in the laboratory by sonication, detergent dialysis, ethanol injection or dilution, French press extrusion, ether infusion, and reverse phase evaporation.
  • MLVs multilamellar lipid vesicles
  • MLVs are candidates for time release drugs because the fluids entrapped between layers are only released as each membrane degrades.
  • UVs may be made small (SUVs) or large (LUVs).
  • liposome formation involves the injection or dropwise addition of lipid in an aqueous buffer.
  • the resulting liposomes are typically heterogenous in size.
  • liposomes are formulated to carry therapeutic agents either contained within the aqueous interior space (water-soluble drugs) or partitioned into the lipid bilayer(s) (water-insoluble drugs). Active agents which have short half-lives in the bloodstream are particularly suited to delivery via liposomes. Many anti-neoplastic agents, for example, are known to have a short half-life in the bloodstream such that their parenteral use is not feasible. However, the use of liposomes for site-specific delivery of active agents via the bloodstream is severely limited by the rapid clearance of liposomes from the blood by cells of the reticuloendothelial system.
  • U.S. Pat No. 5,478,860 which issued to Wheeler et al., on Dec. 26,1995 , and which is incorporated herein by reference, discloses microemulsion compositions for the delivery of hydrophobic compounds. Such compositions have a variety of uses.
  • the hydrophobic compounds are therapeutic agents including drugs.
  • the patent also discloses methods for in vitro and in vivo delivery of hydrophobic compounds to cells.
  • US2004/0142025 restricts the variance between flow rates to less that 50%, more typically to less than about 25% and even more typically less than about 5%.
  • a primary limitation of a "T"-connector or mixing chamber is the requirement of equal momenta of the fluid flows (i.e. solvent and buffer streams) to effect sufficient mixing.
  • MIVM multi-inlet vortex mixer
  • the present invention provides a process for producing lipid nanoparticles (LNPs) encapsulating therapeutic products, said process comprising: a) providing one or more aqueous solutions in one or more reservoirs; b) providing one or more organic solutions in one or more reservoirs, wherein one or more of the organic solutions contains a lipid and wherein one or more of the aqueous solutions and/or one or more of the organic solutions includes therapeutic products; c) mixing said one or more aqueous solutions with said one or more organic solutions in a first mixing region, wherein said one or more aqueous solutions and said one or more organic solutions are introduced into a mixing chamber so as to substantially instantaneously produce a lipid nanoparticle solution containing lipid nanoparticles encapsulating therapeutic products.
  • LNPs lipid nanoparticles
  • the present invention provides a process for producing lipid nanoparticles encapsulating therapeutic products, said process comprising: a) providing one to four aqueous solutions in separate reservoirs; b) providing one to four organic solutions in separate reservoirs, wherein one to four of the organic solutions contains a lipid and wherein one to four of the aqueous solutions and/or one to four of the organic solutions includes therapeutic products; c) mixing said one to four aqueous solutions with said one to four organic solutions in a first mixing region, wherein said one to four aqueous solutions and said one to four organic solutions are introduced into a mixing chamber so as to substantially instantaneously produce a lipid nanoparticle solution containing lipid nanoparticles encapsulating therapeutic products.
  • the present invention provides a process for producing lipid nanoparticles encapsulating therapeutic products, said process comprising: a) providing one to three aqueous solutions in separate reservoirs; b) providing one to three organic solutions in separate reservoirs, wherein one to three of the organic solutions contains a lipid and wherein one to three of the aqueous solutions and/or one to three of the organic solutions includes therapeutic products; c) mixing said one to three aqueous solutions with said one to three organic solutions in a first mixing region, wherein said one to three aqueous solutions and said one to three organic solutions are introduced into a mixing chamber so as to substantially instantaneously produce a lipid nanoparticle solution containing lipid nanoparticles encapsulating therapeutic products.
  • the present invention provides a process for producing lipid nanoparticles encapsulating therapeutic products, said process comprising: a) providing one to two aqueous solutions in separate reservoirs; b) providing one to two organic solutions in separate reservoirs, wherein one to two of the organic solutions contains a lipid and wherein one to two of the aqueous solutions and/or one to two of the organic solutions includes therapeutic products; c) mixing said one to two aqueous solutions with said one to two organic solutions in a first mixing region, wherein said one to two aqueous solutions and said one to two organic solutions are introduced into a mixing chamber so as to substantially instantaneously produce a lipid nanoparticle solution containing lipid nanoparticles encapsulating therapeutic products.
  • the present invention further provides a process for producing lipid nanoparticles encapsulating therapeutic products, said process comprising: a) providing one or more aqueous solutions in one or more reservoirs; b) providing one or more organic solutions in one or more reservoirs, wherein one or more of the organic solutions contains a lipid and wherein one or more of the aqueous solutions and/or one or more of the organic solutions includes therapeutic products; c) mixing said one or more aqueous solutions with said one or more organic solutions in a first mixing region, wherein said first mixing region is a Multi-Inlet Vortex Mixer (MIVM), wherein said one or more aqueous solutions and said one or more organic solutions are introduced tangentially into a mixing chamber within the MIVM so as to substantially instantaneously produce a lipid nanoparticle solution containing lipid nanoparticles encapsulating therapeutic products.
  • MIVM Multi-Inlet Vortex Mixer
  • the process further comprises diluting of said lipid nanoparticle solution in an aqueous buffer immediately after mixing so as to produce a diluted lipid nanoparticle solution.
  • the process further comprises mixing said lipid nanoparticle solution in an aqueous buffer thus introducing the lipid nanoparticle solution into a buffer reservoir containing the aqueous buffer.
  • the buffer reservoir contains an amount of aqueous buffer substantially equal to or greater than the amount of lipid nanoparticle solution introduced thereto.
  • the buffer reservoir is stirred.
  • the process further comprises mixing said lipid nanoparticle solution with the aqueous buffer in a second mixing region.
  • the process further comprises a second mixing region which is a second Multi-Inlet Vortex Mixer (MIVM) wherein the lipid nanoparticle solution and aqueous buffer are introduced tangentially into said second MIVM mixing chamber.
  • MIVM Multi-Inlet Vortex Mixer
  • the process further comprises a lipid nanoparticle solution that has a concentration of about 5% v/v to about 55% v/v organic solvent.
  • the process further comprises a diluted lipid nanoparticle solution that has a concentration of less than about 25% v/v organic solvent.
  • the process further comprises lipid nanoparticles that are less than about 150 nm in diameter.
  • the process further comprises therapeutic products that are nucleic acids.
  • the process further comprises nucleic acids that are siRNAs.
  • Suitable therapeutic products include, but are not limited to, a protein, a nucleic acid, an antisense nucleic acid, a ribozyme, tRNA, snRNA, siRNA (small interfering RNA), miRNA, shRNA, ncRNA, pre-condensed DNA, an aptamer and an antigen.
  • the therapeutic product(s) is/are nucleic acid(s) and/or oligonucleotides.
  • the therapeutic product(s) is/are siRNA(s).
  • nucleic acid refers to a polymer containing at least two nucleotides.
  • Nucleotides contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups (although synthetic nucleic acids may be prepared using nucleotide linkers other than phosphate groups).
  • Bases include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.
  • DNA may be in the form of antisense, plasmid DNA, parts of a plasmid DNA, pre-condensed DNA, product of a polymerase chain reaction (PCR), vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, cbimeric sequences, chromosomal DNA, or derivatives of these groups.
  • PCR polymerase chain reaction
  • vectors Pl, PAC, BAC, YAC, artificial chromosomes
  • expression cassettes cbimeric sequences, chromosomal DNA, or derivatives of these groups.
  • Antisense is a polynucleotide that interferes with the function of DNA and/or RNA. This may result in suppression of expression.
  • Natural nucleic acids have a phosphate backbone
  • artificial nucleic acids may contain other types of backbones and bases. These include PNAs (peptide nucleic acids), phosphothioates, and other variants of the phosphate backbone of native nucleic acids.
  • RNA may be in the form of oligonucleotide RNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, siRNA (small interfering RNA), miRNA, shRNA (shorthairpin RNA), ncRNA (non-coding RNA), aptamers, ribozymes, cbimeric sequences, or derivatives of these groups.
  • tRNA transfer RNA
  • snRNA small nuclear RNA
  • rRNA ribosomal RNA
  • mRNA messenger RNA
  • antisense RNA antisense RNA
  • siRNA small interfering RNA
  • miRNA miRNA
  • shRNA shorthairpin RNA
  • ncRNA non-coding RNA
  • aptamers ribozymes, cbimeric sequences, or derivatives of these groups.
  • siRNA directs the sequence-specific silencing of mRNA through a process known as RNA interference (RNAi).
  • RNAi RNA interference
  • the process occurs in a wide variety of organisms, including mammals and other vertebrates.
  • Methods for preparing and administering siRNA and their use for specifically inactivating gene function are known.
  • siRNA includes modified and unmodified siRNA. Examples and a further discription of siRNA, including modification to siRNAs can be found in WO2009/126933 , which is hereby incorporated by reference.
  • DNA and RNA may be single, double, triple, or quadruple stranded.
  • aqueous solution refers to a composition comprising in whole, or in part, water.
  • organic solution refers to a composition comprising in whole, or in part, an organic solvent.
  • mixing region optionally is any “mixer” known in the art.
  • Particular mixers known in the art include a confined impinging jet (CIJ) mixer or a multi-inlet vortex mixer (MIVM).
  • CIJ confined impinging jet
  • MIVM multi-inlet vortex mixer
  • a “mixer” refers to a device with three or more inlets meeting in a central mixing chamber designed to enhance mixing, and a single outlet.
  • a “mixer” refers to a device with four inlets, meeting in a central mixing chamber, and a single outlet.
  • the "mixer” is a MIVM.
  • lipid refers to a group of organic compounds that are esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. They are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; (3) “derived lipids” such as steroids.
  • amphipathic lipid refers, in part, to any suitable material wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while a hydrophilic portion orients toward the aqueous phase.
  • Amphipathic lipids are usually the major component of lipid nanoparticles. Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, amine, hydroxy and other like groups.
  • Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s).
  • apolar groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s).
  • amphipathic compounds include, but are not limited to, phospholipids, aminolipids and sphingolipids.
  • phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleryl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphospbatidylcholine, dstearoylphosphatidylcholine or dilinoleoylphosphatidylcholine.
  • amphipathic lipids Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols and S-acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipid described above can be mixed with other lipids including triglycerides and sterols.
  • neutral lipid refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.
  • lipids include, for example, diacylphosphatidylcholine, diacylphosphatidyletbanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols.
  • noncationic lipid refers to any neutral lipid as described above as well as anionic lipids.
  • useful noncationic lipids include, for example, distearoylphos-phatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (OPEC), dioleoylphospbatidylglycerol (DOPG), dipahnitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoylolmyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(4-maleimidomethyl)cyelohexane-1-carboxylate (DOPE-teal), dipahnitoyl
  • anionic lipid refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglyeerol (POPG), and other anionic modifying groups joined to neutral lipids.
  • phosphatidylglycerol cardiolipin
  • diacylphosphatidylserine diacylphosphatidic acid
  • N-dodecanoyl phosphatidylethanolamines N-succinyl phosphatidylethanolamines
  • N-glutarylphosphatidylethanolamines
  • cationic lipid refers to any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH.
  • lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC”); N-(2,3dioleyloxy)propyl)-N,N,Ntrimethylammonium chloride (“DOTMA”); N,NdistearylN,N-dimethylammonium bromide ('DDAB”); N-(2,3dioleoyloxy)propyl)-N,N,N-trimethylamntonium chloride (“DODAP”); 3-(N-(N,N-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”) and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydmxyethyl ammoni
  • cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic lipid nanoparticles comprising DOTMA and 1,2dioleoyl-sn-3-phosphoethanolamine ("DOPE"), from GIBCOBRL, Grand Island, N.Y, USA); LIPOFECTAMINE® (commercially available cationic lipid nanoparticles comprising N-(1-(2,3dioleyloxy)propyl)N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (“DOSPA') and (“DOPE”), from (3IBCOBRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioemdecylamidoglycyl carboxyspermine ("DOGS”) in ethanol from Promega Corp., Madison, Wis., USA).
  • LIPOFECTIN® commercially available cationic lipid
  • lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 4-(2,2-diocta-9,12-dienyl-[1,3]dioxolan-4-ylmethyl)-dimethylamine, DLinKDMA ( WO 2009/132131 A1 ), DLin-K-C2-DMA ( WO2010/042877 ), DLin-M-C3-DMA ( WO2010/146740 and/or WO2010/105209 ), 2- ⁇ 4-[(3 ⁇ )-cholest-5-en-3-yloxy]butoxy ⁇ -N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dienlyloxyl]propan-1-amine) (CLinDMA), and the like.
  • DODAP DODAP
  • the lipid nanoparticles of the present invention may comprise bilayer stabilizing component (BSC) such as an ATTA-lipid or a PEG-lipid, such as PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g., WO 05/026372 , PEG coupled to diacylglycerol (PEG-DAG) as described in, e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689 , PEG coupled to dimyristoylglecerol (PEG-DMG) as described in, e.g., Abrams et.
  • BSC bilayer stabilizing component
  • an ATTA-lipid or a PEG-lipid such as PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g., WO 05/026372
  • PEG coupled to diacylglycerol (PEG-DAG) as described in, e.g
  • the BSC is a conjugated lipid that inhibits aggregation of the lipid nanoparticle.
  • the cationic lipid typically comprises from about 2% to about 70%, from about 5% to about 50%, from about 10% to about 45%, from about 20% to about 40%, or from about 30% to about 40% of the total lipid present in said particle.
  • the non-cationic lipid typically comprises from about 5% to about 90%, from about 10% to about 85%, from about 20% to about 80%, from about 30% to about 70%, from about 40% to about 60% or about 48% of the total lipid present in said particle.
  • the PEG-lipid conjugate typically comprises from about 0.5% to about 20%, from about 1.5% to about 18%, from about 4% to about 15%, from about 5% to about 12%, or about 2% of the total lipid present in said particle.
  • the nucleic acid-lipid particles of the present invention may further comprise cholesterol.
  • the cholesterol typically comprises from about 0% to about 10%, about 2% to about 10%, about 10% to about 60%, from about 12% to about 58%, from about 20% to about 55%, or about 48% of the total lipid present in said particle. It will be readily apparent to one of skill in the art that the proportions of the components of the nucleic acid-lipid particles may be varied.
  • the nucleic acid to lipid ratios (mass/mass ratios) in a formed nucleic acid-lipid particle will range from about 0.01 to about 0.3.
  • the ratio of the starting materials also falls within this range
  • Lipid Nanoparticle refers to any lipid composition that can be used to deliver a therapeutic product, preferably siRNAs or an siRNA, including, but not limited to, liposomes or vesicles, wherein an aqueous volume is encapsulated by amphipathic lipid bilayers ( i.e. single; unilamellar or multiple; multilamellar), or wherein the lipids coat an interior comprising a therapeutic product, or lipid aggregates or micelles, wherein the lipid encapsulated therapeutic product is contained within a relatively disordered lipid mixture.
  • a therapeutic product preferably siRNAs or an siRNA
  • lipid encapsulated can refer to a lipid formulation which provides a therapeutic product with full encapsulation, partial encapsulation, or both.
  • LNP refers to a lipid nanoparticle
  • the present invention provides processes for making lipid nanoparticles.
  • the processes can be used to make lipid nanoparticles possessing a wide range of lipid components including, but not limited to, cationic lipids, anionic lipids, neutral lipids, polyethylene glycol (PEG) lipids, hydrophilic polymer lipids, fusogenic lipids and sterols.
  • Hydrophobic actives can be incorporated into the organic solvent (e.g., ethanol) with the lipid, and nucleic acid and hydrophilic actives can be added to an aqueous component.
  • the processes of the present invention can be used in preparing microemulsions where a lipid monolayer surrounds an oil-based core.
  • the processes and apparatus are used in preparing lipid nanoparticles, wherein a therapeutic agent is encapsulated within a lipid nanoparticle coincident with lipid nanoparticle formation.
  • FIG. 1 is an example of a representative flow chart 100 of a method of the present invention. This flow chart is merely an illustration and should not limit the scope of the claims herein. One of ordinary skill in the art will recognize other variations, modifications, and alternatives.
  • the present method provides a lipid solution 110 such as a clinical grade lipid synthesized under Good Manufacturing Practice (GMP), which is thereafter solubilized in an organic solution 120 (e.g., ethanol).
  • a therapeutic product e.g., a therapeutic active agent such as nucleic acid 112 or other agent
  • GMP Good Manufacturing Practice
  • a therapeutic agent solution e.g., nucleic acids
  • a buffer e.g., citrate
  • lipid nanoparticle formulation 130 also referred to herein as "lipid nanoparticle suspension” or "lipid nanoparticle solution”
  • the therapeutic agent is entrapped in the lipid nanoparticle substantially coincident with formation of the lipid nanoparticle.
  • an electrostatic interaction between the negatively charged nucleic acid and positively charged cationic lipid brings about encapsulation.
  • a titratable cationic lipid is used, for example, poor nucleic acid encapsulation efficiencies may be achieved at higher pH approaching or exceeding the cationic lipids pKa.
  • the processes and apparatus of the present invention are equally applicable to active entrapment or loading of the lipid nanoparticles after formation of the lipid nanoparticle.
  • the lipid nanoparticle solution is substantially immediately mixed with a buffer solution 140 to dilute the nanoparticle solution (i.e., suspension of lipid nanoparticles).
  • the action of continuously introducing lipid and buffer solutions into a mixing environment causes a continuous dilution of the lipid solution with the buffer solution, thereby producing a lipid nanoparticle substantially instantaneously upon mixing.
  • a continuous dilution of the lipid solution with the buffer solution e.g., mixing the lipid nanoparticle suspension with buffer
  • immediate dilution helps prevent lipid nanoparticle particle sizes from increasing as would typically be the case if the lipid nanoparticle suspension is allowed to sit for an extended period of time, e.g., minutes or hours.
  • immediate dilution further enhances lipid nanoparticle homogeneity especially where siRNA is the encapsulated therapeutic agent.
  • the phrase "continuously diluting a lipid solution with a buffer solution” generally means that the lipid solution is diluted sufficiently rapidly in a hydration process with sufficient force to effectuate lipid nanoparticle generation.
  • the organic lipid solution typically includes an organic solvent, such as a lower alcohol.
  • the lipid nanoparticles are immediately diluted 140 with a buffer (e.g., citrate) to increase nucleic acid (e.g., plasmid or siRNA) entrapment and maintain particle size.
  • a buffer e.g., citrate
  • nucleic acid e.g., plasmid or siRNA
  • Such dilution may be by way of immediate introduction of the lipid nanoparticle solution into a controlled amount of buffer solution, or by mixing the lipid nanoparticle solution with a controlled flow rate of buffer in a second mixing region.
  • Dilution can also be effected coincident with lipid nanoparticle formation 130 at initial introduction of organic lipid and buffer solutions into a mixing environment
  • organic lipid and buffer solutions are introduced into a mixing environment at substantially non-equal flow-rates such that resulting lipid nanoparticle solution contains a volumetric excess of dilution buffer.
  • sample concentration 160 Before sample concentration 160, free therapeutic agent (e.g., nucleic acid) is removed by using, for example, an anion exchange cartridge 150. Further, by using an ultrafiltration step 170 to remove the organic solution, the sample is concentrated (e.g., to about 0.9 mg/mL nucleic acid), the organic solution (e.g. alcohol) is removed, and the buffer is replaced with a substitute buffer (e.g., with a saline buffer) 180. Thereafter, the sample is filtered 190 and filled in vials 195. The process will now be discussed in more detail herein below using the steps as set forth in FIG. 1 .
  • a substitute buffer e.g., with a saline buffer
  • the lipid nanoparticle produced according to the processes of the present invention include nucleic acid lipid nanoparticle (i.e., LNP) formulations.
  • LNP nucleic acid lipid nanoparticle
  • lipid nanoparticles include, but are not limited to, single bilayer lipid vesicles known as unilamellar lipid vesicles which can be made small (SUVs) or large (LUVs), as well as multilamellar lipid vesicles (MLVs).
  • Further vesicles include, micelles, lipid nucleic acid particles, virosomes, and the like. Those of skill in the art will know of other lipid vesicles for which the processes and apparatus of the present invention will be suitable.
  • the preferred size for lipid nanoparticles made in accordance with the present processes and apparatus are between about 25-200 nm in diameter.
  • the lipid nanoparticle preparation has a size distribution in which the mean size (e.g., diameter) is about 70 nm to about 200 nm, and more typically the mean size is about 100 nm or less.
  • the lipid nanoparticle formulation (e.g., LNP formulation) of the present invention includes four lipid components; a phospholipid; cholesterol; a PEG-lipid; and a cationic lipid.
  • the phospholipid is DSPC
  • the PEG-lipid is PEG-S-DMG
  • the cationic lipid is CLinDMA or DLinDMA.
  • the molar composition is about 60:38:2 CLinDMA:choesterol:PEG-DMG.
  • the LNP formulation is 40:48:10:2 DLinDMA:cholesterol:DSPC:PEG-DMG.
  • the organic solvent concentration wherein the lipids are solubilized is about 45% v/v to about 100% v/v.
  • the organic solvent is a lower alcohol. Suitable lower alcohols include, but are not limited to, methanol, ethanol, propanol, butane, pentanol, their isomers and combinations thereof.
  • the solvent is ethanol with a volume of about 50-90% v/v.
  • the lipids occupy a volume of about 1 mL/g to about 5 mL/g.
  • the lipids are solubilized 120 using for example, an overhead stirrer at a suitable temperature.
  • the total lipid concentration of the solution is about 15. mg/mL (6.8 mg/mL for LNP formulation).
  • the therapeutic agent e.g., nucleic acid
  • the aqueous solution e.g., buffer
  • the final concentration is about 0.8 mg/mL in citrate buffer, with a pH of about 4-6.
  • the volume of the nucleic acid solution is the same as the alcohol-lipid solution.
  • the buffer solution need not be acidic when using the direct dilution approaches of the present invention, e.g, the pH of the buffer solution can be 7.0 or higher.
  • the preparation of the therapeutic agent (e.g., nucleic acid) solutions performed in a jacketed stainless steel vessel with an overhead mixer. The sample does not need to be heated to be prepared, although in certain instances it is at the same temperature as the lipid solution prior to lipid nanoparticle formation.
  • the solutions e.g., lipid solution 120 and aqueous therapeutic agent (e.g., nucleic acid) solution 115
  • they are mixed together 130 using, for example, a peristaltic pump mixer or a pulseless gear pump.
  • the solutions are pumped at substantially equal flow rates into a mixing environment.
  • the mixing environment includes a Multi-Inlet Vortex Mixer (MIVM) or mixing chamber.
  • MIVM Multi-Inlet Vortex Mixer
  • the solutions are pumped at substantially equal flow rates into the MIVM mixing environment.
  • the solutions are pumped at substantially unequal flow rates.
  • Lipid nanoparticles are formed when an organic solution including dissolved lipid and an aqueous solution (e.g., buffer) are simultaneously and continuously mixed.
  • an aqueous solution e.g., buffer
  • the organic lipid solution undergoes a continuous, sequential stepwise dilution to substantially instantaneously produce a lipid nanoparticle solution.
  • the pump mechanism(s) can be configured to provide equivalent or different flow rates of the lipid and aqueous solutions into the mixing environment.
  • the apparatus for mixing of lipid and the aqueous solutions taught herein provides for formation of lipid nanoparticles under conditions where the composition of buffer and organic solutions can be changed over a wide range, without loss of mixing efficiency.
  • substantially equal momenta (i.e. flow rates) of the fluid flows are required to effect sufficient mixing
  • in the MIVM momentum (i.e. flow rate) from each stream contributes independently to drive micromixing in the mixing chamber. Therefore, it is possible to have one or more streams at high volumetric flow rate and another stream at a lower flow rate and still achieve good micromixing.
  • the ratio of organic to buffer solutions at initial mixing can be advantageously manipulated to effect better control over lipid particle properties (e.g. particle size and particle size stability).
  • the processes and apparatus for mixing of the lipid solution and the aqueous solutions taught herein provides for encapsulation of therapeutic agent in the formed lipid nanoparticle substantially coincident with lipid nanoparticle formation with an encapsulation efficiency of up to about 90%. Further processing steps as discussed herein can be used to further refine the encapsulation efficiency and concentration if desired.
  • lipid nanoparticles form when lipids dissolved in an organic solvent (e.g., ethanol) are diluted in a stepwise manner by mixing with an aqueous solution (e.g., buffer).
  • an aqueous solution e.g., buffer
  • This controlled stepwise dilution is achieved by mixing the aqueous and lipid streams together in a confined volume, such as in a MIVM, and thereafter diluting in a buffer solution.
  • the resultant lipid, solvent and solute concentrations can be kept constant throughout the nanoparticle formation process if desired.
  • lipid nanoparticles are formed having a mean diameter of less than about 150 nm, e.g., about 100 nm or less, which advantageously do not require further size reduction by high-energy processes such as membrane extrusion, sonication or microfluidization.
  • a lipid nanoparticle solution is prepared by a two-stage step-wise dilution.
  • lipid nanoparticles are formed in a high organic solvent (e.g., ethanol) environment (e.g., about 35% v/v to about 55% v/v organic solvent).
  • a second (e.g. dilution) step by lowering the organic solvent (e.g., ethanol) concentration to less than or equal to about 25% v/v such as about 17% v/v to about 25% v/v, in a stepwise manner.
  • organic solvent e.g., ethanol
  • Such dilution may be by way of immediate introduction of the lipid nanoparticle solution into a controlled amount of buffer solution, or by mixing the lipid nanoparticle solution with a controlled flow rate of buffer in a second mixing region.
  • the therapeutic agent is encapsulated coincident with lipid nanoparticle formation.
  • lipids are initially dissolved in an organic solvent (e.g., ethanol) environment of about 70% v/v to about 100% v/v, more typically about 65% v/v to about 90% v/v, and most typically about 80% v/v to about 100% v/v (A).
  • an organic solvent e.g., ethanol
  • the lipid solution is diluted stepwise by mixing with an aqueous solution resulting in the formation of lipid nanoparticles at an organic solvent (e.g., ethanol) concentration of about 35% v/v to about 55% v/v, more typically about 33% v/v to about 45% v/v, and most typically about 40% v/v to about 50% v/v (B).
  • organic solvent e.g., ethanol
  • lipid nanoparticles such as LNPs can be further stabilized by an additional stepwise dilution of the nanoparticles to an alcohol concentration of less than or equal to about 25%, preferably between about 19-25% (C).
  • the additional sequential dilution (C) is performed substantially immediately after formation of the lipid nanoparticles. For example, it is advantageous that less than I minute elapse between lipid nanoparticle solution formation and dilution (C), more advantageously less than 10 seconds and even more advantageously less than a second or two.
  • a lipid nanoparticle solution is prepared by a one-stage step-wise process in which lipid nanoparticle formation occur coincidentally with dilution.
  • lipids are initially dissolved in an organic solvent (e.g., ethanol) environment of about 70% v/v to about 100% v/v, more typically about 65% v/v to about 90% v/v, and most typically about 80% v/v to about 100% v/v (A).
  • an organic solvent e.g., ethanol
  • the lipid solution is diluted stepwise by mixing with an aqueous solution resulting in the formation of nanoparticles at an organic solvent (e.g., ethanol) concentration of about 5% v/v to about 35% v/v, more typically about 15% v/v to about 30% v/v, and most typically about 10% v/v to about 25% v/v (C).
  • an organic solvent e.g., ethanol
  • concentration of about 5% v/v to about 35% v/v, more typically about 15% v/v to about 30% v/v, and most typically about 10% v/v to about 25% v/v (C).
  • the lipid nanoparticles are formed at a rate of 40 to about 400 mL/min
  • the lipid concentration is about 1-12 mg/mL and the therapeutic agent (e.g., nucleic acid) concentration is about 0.05-1 mg/mL.
  • the lipid concentration is about 3.4 mg/ml, and the therapeutic agent (e.g., nucleic acid) concentration is about 0.8 mg/mL to give a lipid--nucleic acid ratio of about 4.
  • the buffer concentration is about 1-25 mM and the organic solvent (e.g., alcohol) concentration is about 5% v/v to about 95% v/v.
  • the buffer concentration is about 25 mM and the organic solvent (e.g., alcohol) concentration is about 20% v/v to about 50% v/v.
  • the degree of therapeutic agent (e.g., nucleic acid) encapsulation is enhanced and particle size maintained, and even reduced, by immediate diluting 140 the lipid nanoparticle solution prior to removal of free nucleic acid.
  • the therapeutic agent entrapment is at about 30-60%, it can be increased to about 80-90% following dilution step 140.
  • the lipid nanoparticle formulation is diluted to about 10% v/v to about 40% v/v, preferably about 20% alcohol, by mixing with an aqueous solution such as a buffer (e.g., 1:1 with 20 mM citrate buffer, 300 mM NaCl, pH 6.0). The diluted sample is then optionally allowed to incubate at room temperature.
  • a buffer e.g., 1:1 with 20 mM citrate buffer, 300 mM NaCl, pH 6.0.
  • Dilution may be effected by way of immediate introduction of the lipid nanoparticle solution into a controlled amount of buffer solution, or by mixing the lipid nanoparticle solution with a controlled flow rate of buffer in a second mixing region.
  • dilution can be effected coincident with lipid nanoparticle formation 130, upon initial introduction of organic lipid and buffer solutions into the mixing environment.
  • organic lipid and buffer solutions are introduced into a mixing environment at substantially non-equal flow-rates such that the resulting lipid nanoparticle solution contains a volumetric excess of dilution buffer.
  • the diluted sample is then optionally allowed to incubate at room temperature.
  • the therapeutic agent e.g., nucleic acid
  • the lipid nanoparticle e.g., LNP
  • anion exchange chromatography is used.
  • the use of an anion exchange resin results in a high dynamic nucleic acid removal capacity, is capable of single use, may be pre-sterilized and validated, and is fully scaleable.
  • the method results in removal of free therapeutic agent (e.g., nucleic acid).
  • the volume of sample after chromatography is unchanged, and the therapeutic agent (e.g., nucleic acid) and lipid concentrations are about 0.3 and 1.7 mg/mL, respectively. At this point, the sample can be assayed for encapsulated therapeutic agent.
  • the lipid nanoparticle solution is optionally concentrated about 5-50 fold, preferably 10-20 fold, using for example, ultrafiltration 160 (e.g., tangential flow dialysis).
  • the sample is transferred to a feed reservoir of an ultrafiltration system and the buffer is removed.
  • the buffer can be removed using various processes, such as by ultrafiltration.
  • buffer is removed using cartridges packed with polysulfone hollow fibers, for example, having internal diameters of about 0.5 mm to about 1.0 mm and a 30,000 nominal molecular weight cut-off (NMWC). Hollow fibers with about a 1,000 MWCO to about a 750,000 MWCO may also be used.
  • the lipid nanoparticles are retained within the hollow fibers; and recirculated while the solvent and small molecules are removed from the formulation by passing through the pores of the hollow fibers. In this procedure, the filtrate is known as the permeate solution.
  • the therapeutic agent e.g., nucleic acid
  • lipid concentrations can increase to about 2 and 60 mg/mL, respectively.
  • the organic solvent e.g., alcohol
  • the organic solvent:lipid ratio decreases about 50 fold.
  • the concentrated formulation is diafiltered against about 5-20 volumes, preferably about 10 volumes, of aqueous solution (e.g., citrate buffer pH 4.0 (25 mM citrate, 100 mM NaCl) to remove the alcohol 170.
  • aqueous solution e.g., citrate buffer pH 4.0 (25 mM citrate, 100 mM NaCl)
  • a neutral buffer or a sugar-based buffer may also be used.
  • the organic solvent (e.g., alcohol) concentration at the completion of step 170 is less than about 1%.
  • Lipid and therapeutic agent (e.g., nucleic acid) concentrations remain unchanged and the level of therapeutic agent entrapment also remains constant.
  • the aqueous solution e.g., buffer
  • the aqueous solution e.g., buffer
  • diafiltration against another buffer 180 e.g., against 10 volumes of saline 150 mM NaCl with 10 mM HEPES or Phosphate pH 7.4
  • buffers e.g., neutral, sugar-based, etc.
  • the ratio of concentrations of lipid to therapeutic agent e.g., nucleic acid
  • sample yield can be improved by rinsing the cartridge with buffer at about 10% volume of the concentrated sample. In certain aspects, this rinse is then added to the concentrated sample.
  • sterile filtration 190 of the sample at lipid concentrations of about 12-120 mg/mL can optionally be performed.
  • filtration is conducted at pressures below about 40 psi, using a capsule filter and a pressurized dispensing vessel with a heating jacket. Heating the sample slightly can improve the ease of filtration.
  • the sterile fill step 195 is performed using similar processes as for conventional liposomal formulations.
  • the processes of the present invention result in about 50-60% of the input therapeutic agent (e.g., nucleic acid) in the final product.
  • the therapeutic agent to lipid ratio of the final product is approximately 0.01 to 0.2.
  • lipid-based drug formulations and compositions of the present invention are useful for the systemic or local delivery of therapeutic products and are also useful in diagnostic assays.
  • therapeutic product is preferably incorporated into the lipid nanoparticle during formation of the nanoparticle
  • hydrophobic actives can be incorporated into the organic solvent with the lipid
  • nucleic acid and hydrophilic therapeutic products can be added to the aqueous component.
  • the therapeutic products includes one of a protein, a nucleic acid, an antisense nucleic acid, ribozymes, tRNA, snRNA, siRNA, miRNA sbRNA, mRNA, pre-condensed DNA, an aptamer, an antigen and combinations thereof.
  • the therapeutic product is nucleic acid.
  • the therapeutic product is a siRNA.
  • therapeutic product is incorporated into the organic lipid component.
  • the lipid nanoparticles of the present invention can be loaded with one or more therapeutic products after formation of the nanoparticle.
  • the therapeutic products which are administered using the present invention can be any of a variety of drugs which are selected to be an appropriate treatment for the disease to be treated.
  • the drug is an siRNA.
  • the present invention provides systems and apparatus for carrying out the processes of the present invention.
  • FIGS. 3A and 3B show examples of an apparatus 300 and apparatus 302, respectively, according to two embodiments of the present invention. These schematics are merely illustrations and should not limit the scope of the claims herein. One of ordinary skill in the art will recognize other variations, modifications, and alternatives.
  • apparatus 300 includes two reservoirs, an aqueous solution reservoir 305 and an organic solution reservoir 310, for holding aqueous solution and organic solution, respectively.
  • Apparatus 302 includes four reservoirs, including an aqueous solution reservoir 305 and an organic solution reservoir 310, for holding aqueous solution and organic solution, respectively.
  • the third and fourth reservoirs 315 and 320 are used for holding either aqueous solution, or organic solution, or a combination thereof.
  • the lipid nanoparticle formulations are prepared rapidly, at low pressure (e.g., ⁇ 10 psi) and the apparatus and processes of the present invention are fully scaleable (e.g., 0.5 mL-5000 L). At a 1-L scale, lipid nanoparticles are formed at about 0.4-1.7 L/min.
  • the apparatus does not use static mixers nor specialized extrusion equipment.
  • FIG. 4 shows a Multi-Inlet Vortex Mixer (MIVM) according to one embodiment.
  • the mixing chamber 330 includes, in one embodiment, a mixing chamber, having optional hose barbs, wherein fluid lines impact each other tangentially (and not at 180° or angles thereabout, as per "T"-connector of Impinging Jets-type mixing geometries).
  • lipid nanoparticles of well defined and reproducible mean diameters are prepared using substantially equal flow rates of the flow lines.
  • lipid nanoparticles of well defined and reproducible mean diameters are prepared using substantially non-equal flow rates of the fluid lines. Examples of flow rates are shown and discussed in more detail in the Example section (below).
  • the apparatus for mixing of lipid and the aqueous solutions taught herein provides for formation of lipid nanoparticles under conditions where the concentration of buffer and organic solutions can be changed over a wide range, without loss of mixing efficiency.
  • substantially equal momenta (i.e. flow rates) of the fluid flows are required to effect sufficient mixing
  • in the MIVM momentum (i.e. flow rate) from each stream contributes independently to drive micromixing in the mixing chamber. Therefore, it is possible to have one or more streams at high volumetric flow rate and another stream at a lower flow rate and still achieve good micromixing.
  • the ratio of organic to buffer solutions at initial mixing can be advantageously manipulated to effect better control over lipid particle properties (e.g. particle size, size stability, nucleic acid encapsulation, lipid rearrangement, etc.).
  • the present invention advantageously permits the formation of LNPs at solvent concentrations as low as 5% v/v, more typically 10 % v/v, and even more typically 25% v/v in a single mixing step.
  • therapeutic agent e.g., nucleic acid
  • encapsulation is enhanced and particle size maintained, and even reduced, relative to alternative dilution strategies (e.g. dilution 140 of the lipid nanoparticle suspension by way of introduction of the lipid nanoparticle solution into a controlled amount of buffer solution, or by mixing the lipid nanoparticle solution with a controlled flow rate of buffer in a second mixing region).
  • the processes and apparatus of the present invention further provide operational flexibility in the formulation of lipid nanoparticles and lipid nanoparticles encapsulating therapeutic agents.
  • the solubility of amphipathic lipids or lipophilic therapeutic agents is expected to decrease with addition of aqueous phase.
  • the concentrations of lipids or lipophilic therapeutic agents in organic fluid flow to the mixing chamber are inherently limited by the aqueous buffer concentration in said organic fluid flow, which is in turn dictated by the solvent composition desired to effect lipid nanoparticle formation.
  • amphipathic lipids or lipophilic therapeutic agents can be solubilized within individual non-aqueous organic solutions, and mixed freely against aqueous buffer to effect lipid nanoparticle formation. In this capacity, the concentrations of constituent components in the inlet streams can be maximized.
  • the apparatus described further permits the solubilization of either non-compatible or multiple lipids or therapeutic agents in individual organic or aqueous streams.
  • Mixing of the fluid components can be driven using, for example, a peristaltic pump, a positive displacement pump, a pulseless gear pump, by pressurizing both the lipid-organic solution and buffer vessels or by a combination of two or more of these and/or other pump mechanisms.
  • digitally controlled syringe pumps Hard Apparatus, PHD 2000 programmable
  • teflon tubing available from Upchurch Scientific
  • Lipid nanoparticles are typically formed at room temperature, but lipid nanoparticles may be formed at elevated temperatures according to the present invention.
  • the processes and apparatus of the present invention can formulate a lipid nanoparticle by mixing lipid in an alcohol with water.
  • the processes and apparatus of the present invention form lipid nanoparticles that are less than about 100 nm in diameter.
  • nucleic acid such as LNPs
  • the ratio of nucleic acid to cationic lipid and counter ions can be optimized.
  • nucleic acid such as LNPs
  • the level of NA encapsulation is advantageously increased by immediately diluting this initial LNP formulation.
  • the processes and apparatus of the present invention provide an encapsulation efficiency, upon mixing the solutions (with therapeutic agent in one of the solution components) in the mixing environment, of up to about 90%.
  • Three embodiments of dilution, e.g., direct dilution, are shown in FIG 5 .
  • the lipid nanoparticle solution formed in mixing region 330 is immediately and directly introduced into a collection vessel 340 containing a controlled amount of dilution buffer.
  • vessel 340 includes one or more elements configured to stir the contents of vessel 340 to facilitate dilution.
  • the amount of dilution buffer present in vessel 340 is substantially equal to the volume of lipid nanoparticle solution introduced thereto.
  • lipid nanoparticle solution in 45% ethanol when introduced into vessel 340 containing an equal volume of ethanol will advantageously yield smaller particles in 22.5% ethanol.
  • reservoirs 345 containing dilution buffer are fluidly coupled to a second mixing region 350.
  • the lipid nanoparticle solution formed in mixing region 330 is immediately and directly mixed with dilution buffer in the second mixing region 350.
  • mixing region 350 includes a MIVM arranged so that the lipid nanoparticle solution and the dilution buffer flows meet tangentially, however, connectors providing other mixing angles, such as "T"-connector, where fluid flows meet at 180° relative to each other can also be used.
  • a pump mechanism delivers a controllable flow of buffer to mixing region 350.
  • the flow rate of dilution buffer provided to mixing region 350 is controlled to be substantially equal to the flow rate of lipid nanoparticle solution introduced thereto from mixing region 330.
  • This embodiment advantageously allows for more control of the flow of dilution buffer mixing with the lipid nanoparticle solution in the second mixing region 350, and therefore also the concentration of lipid nanoparticle solution in buffer throughout the second mixing process.
  • Such control of the dilution buffer flow rate advantageously allows for small particle size formation at reduced concentrations. See, e.g., the Examples section below.
  • lipid nanoparticle formation occurs coincidentally with dilution.
  • organic lipid and buffer solutions are introduced into the mixing environment (e.g., four-stream MIVM) at substantially non-equal flow-rates such that the resulting lipid nanoparticle solution contains a volumetric excess of dilution buffer.
  • lipid nanoparticle producing apparatus 300 and 302 of the present invention further includes a temperature control mechanism (not shown) for controlling the temperature of the reservoirs 305 and 310.
  • a temperature control mechanism for controlling the temperature of the reservoirs 305 and 310.
  • fluid from the first reservoir 305 and the second reservoirs 310 flows into mixing chamber 330 simultaneously at separate apertures.
  • Apparatus 300 and 302 further includes a collection reservoir 340 downstream of the mixing chamber 330 for lipid nanoparticle collection.
  • apparatus 300 and 302 further include storage vessels upstream of any or all of the reservoirs 305, 310, 315, and 320.
  • any or all of the reservoirs 305, 310, 315 , and 320 can include jacketed stainless steel vessels equipped with an overhead mixer.
  • the present invention provides an apparatus having an ultrafiltration system (not shown) for carrying out the processes of the present invention.
  • apparatus includes a plurality of reservoirs and is equipped with an ultrafiltration system.
  • An aqueous solution reservoir (not shown) and an organic solution reservoir (not shown) each have upstream preparation nanoparticles (not shown), respectively.
  • the collection vessel (not shown) is in fluid communication with the flow ultrafiltration system.
  • ultrafiltration is used to concentrate LNP samples and then remove organic solvent (e.g., ethanol) from the formulation by buffer replacement.
  • the diluted LNP solutions are transferred to the feed reservoir of the ultrafiltration system.
  • Concentration is performed by removing buffer and organic solution (e.g. ethanol) using, for example, cross flow cartridges 465 packed with polysulfone hollow fibers that possess internal diameters of about 0.5 mm to about 1.0 mm and about 1,000 to about 750,000 molecular weight cut-off (MWCO).
  • the LNP are retained within the hollow fibers and re-circulated, whereas the organic solution (e.g., ethanol) and buffer components are removed from the formulation by passing through the pores of these hollow fibers. This filtrate is known as the permeate solution and is discarded.
  • the buffer in which the LNPs are suspended may be removed by ultrafiltration and replaced by an equal volume of the final buffer. Ultrafiltration can be replaced with other methods such as conventional dialysis.
  • This Example illustrates the use of one process of the present invention to make Lipid Nanoparticles (LNPs) which encapsulate siRNA as therapeutic product.
  • the Example also illustrates the variation of a process parameter (e.g., inlet flow rates) according to one embodiment of the present invention.
  • Oligonucleotide synthesis is well known in the art. (See US patent applications: US 2006/0083780 , US 2006/0240554 , US 2008/0020058 , US 2009/0263407 and US 2009/0285881 and PCT patent applications: WO 2009/086558 , WO2009/127060 , WO2009/132131 , WO2010/042877 , WO2010/054384 , WO2010/054401 , WO2010/054405 and WO2010/054406 ).
  • the siRNAs disclosed and utilized in the Examples were synthesized via standard solid phase procedures.
  • LNPs were prepared as follows. siRNA to luciferase (See Abrams et al. Mol. Therapy (2010) 18(1):171-180 ; Tao et al. Mol. Therapy (2010) 18(9):1657-1666 ; and Morrisey et al. Nat. Biotechnology (2005) 23:1002-1007 ), target strand sequence ATAAGGCTATGAAGAGATA, was dissolved in citrate buffer (25 mM, 100 mM NaCl, pH 3.8) at 47 ⁇ M.
  • Lipids (CLinDMA, PEG-DMG) and cholesterol were solubilized in ethanol at a relative molar ratio of 60:38:2 (CLinDMA:Cholesterol:PEG-DMG) and a total lipid concentration of 6.7 mg/mL.
  • the organic solution was mixed with buffer solution using a two-stream MIVM. Flow rates of inlet solution streams were varied from about 12 mL/min to 70 mL/min per stream (about 24 mL/min to 140 mL/min total). Mixing yielded a particle suspension, wherein the ethanol concentration after mixing was 50% v/v. The obtained suspension was aged at room temperature for 12-18 hours.
  • LNPs varying the total flow rate of inlet solutions to the MIVM had a significant impact on the size of formed LNPs.
  • LNPs possessed mean diameters of 150 nm +/- 20 nm (see, FIG. 6A ) and narrow particle size distributions (PDI; see, FIG. 6B ).
  • Further increases in total flow rate e.g., from about 60 mL/min to about 150 mL/min
  • LNPs could also be prepared, although with larger particle diameters.
  • siRNA encapsulation efficiencies greater than 95% (see, FIG. 6A ). It should be appreciated that other conditions and parameters may be used and those used herein are merely exemplary.
  • Re The Reynold's number
  • D i characteristic length
  • This Example illustrates the use of one process of the present invention to make Lipid Nanoparticles (LNPs) which encapsulate siRNA as therapeutic product
  • the Example also illustrates the variation of a process parameter (e.g., ethanol concentration) according to one embodiment of the present invention.
  • a process parameter e.g., ethanol concentration
  • LNPs were prepared as follows. siRNA to luciferase was dissolved in citrate buffer (25 mM, 100 mM NaCl, pH 3.8) at 47 ⁇ M. Lipids (CLinDMA, PEG-DMG) and cholesterol were solubilized in ethanol at a relative molar ratio of 60:38:2 (CLinDMA:Cholesterol:PEG-DMG) and a total lipid concentration of 6.7 mg/mL. The organic solution was mixed with buffer solution using either a two-stream MIVM or a four-stream MIVM. The solvent concentration (e.g., ethanol:buffer volumetric ratio) was changed by changing the feed flow rates to the MIVM.
  • citrate buffer 25 mM, 100 mM NaCl, pH 3.8
  • Lipids CLinDMA, PEG-DMG
  • cholesterol were solubilized in ethanol at a relative molar ratio of 60:38:2 (CLinDMA:Cholesterol:PEG-DMG) and
  • the flow rate of ethanol solution was varied between 22 mL/min and 11.8 mL/min while keeping the buffer solution flow rate constant at 22 mL/min. Mixing under these process conditions yielded particle suspensions, wherein the ethanol concentration after mixing was varied from 50% v/v to 35% v/v.
  • a four-stream MIVM was used. In these instances, two additional citrate buffer (25 mM, 100 mM NaCl, pH 3.8) streams were used to reduce the ethanol concentration from 35% v/v to 10% v/v.
  • LNP suspensions were diluted immediately following mixing (1:5 v/v LNP suspension to citrate buffer; 25 mM, 100 mM NaCl, pH 3.8), and dialyzed against phosphate buffered saline (PBS, pH 7) using a 6K-8K MWCO Spectra/Pore dialysis membrane. Dialysis was performed to exhange citrate buffer and to remove ethanol. Free (unencapsulated) siRNA does not pass through the dialysis membrane. Following dialysis, LNPs were characterized for particle size and siRNA encapsulation efficiency.
  • LNPs were formed under all ethanol concentrations examined in this embodiment (i.e., ranging from 10% v/v to 50% v/v). The percent ethanol at mixing was found to have a significant impact on particle size (see, FIG. 7 inset). For example, LNPs mixed in 50% v/v ethanol and diluted immediately thereafter with excess buffer, possessed a mean diameter of 120 nm. Reducing the ethanol concentration at mixing to 25% v/v, effectively reduced the mean diameter of LNPs to 70 nm. Additional lowering of ethanol concentration at mixing to 10% v/v yielded no further decreases in LNP diameters. Reductions in LNP size are attributed to increase in lipid supersaturation with decreasing ethanol content. Thus, the ethanol concentration at mixing was found to be a critical process parameter dictating LNP formation, and could thus be manipulated rationally to effect desired LNP size.
  • Varying the ethanol concentration at mixing according to the processes described in this embodiment additionally demonstrates the ability to effectively dilute (and stabilize) LNPs coincident with their formation (i.e. in a single mixing step). While other processes for dilution of lipsome suspensions have been described herein (see, FIG. 5A and 5B ), they all require multiple steps. In the processes described in this Example, mixing of the organic lipid solution with a volumetric excess of aqueous buffer (i.e., non-equal flow rates of organic and aqueous solutions to MIVM), effectively permits the organic solution to undergo a stepwise dilution to produce a lipid nanoparticle, while simultaneously being diluted.
  • a volumetric excess of aqueous buffer i.e., non-equal flow rates of organic and aqueous solutions to MIVM
  • the ethanol concentration was also found to have an impact on the encapsulation efficiency of siRNA (see, FIG. 7 ). Encapsulation efficiencies exceeding 87% were found for LNPs prepared with ethanol concentrations of between 35% v/v and 50% v/v at mixing. Lowering the ethanol concentration at mixing yielded LNPs with somewhat lower encapsulation efficiencies (e.g., 65% and 78% for 10% v/v and 25% v/v ethanol, respectively). It is hypothesized that at the higher ethanol concentrations, the rearrangement of lipid monomers into bilayers proceeds in a more orderly fashion compared to LNPs that are formed by dilution at lower ethanol concentrations.
  • nucleic acid encapsulation occurs within a range of ethanol concentrations between 10% v/v to about 50% v/v, but preferably between about 25% v/v to 35% v/v ethanol.
  • LNP formation at 25% v/v ethanol permits optimization of both LNP size (e.g. ⁇ 150 nm, more preferably ⁇ 100 nm) and encapsulation efficiency (> 75%).
  • This Example illustrates the use of non-alcohol organic solvents to generate LNPs according to one embodiment of the present invention.
  • LNPs were prepared as follows. siRNA to luciferase was dissolved in citrate buffer (25 mM, 100 mM NaCl, pH 3.8) at 47 ⁇ M. Lipids (CLinDMA, PEG-DMG) and cholesterol were solubilized in tetrahydrofuran (THF) at a relative molar ratio of 60:38:2 (CLinDMA:Cholesterol:PEG-DMG) and a total lipid concentration of 6.7 mg/mL. To generate LNPs, the organic lipid solution was mixed with buffer solution using a four-stream MIVM.
  • LNPs with diameters below 80 nm, and more preferably below 55 nm could be produced (see, FIG. 8 ). These LNP suspensions also possessed narrow particle size distributions (see, FIG. 8 ). Both reductions in LNP size and narrowing of size distributions with decreasing THF concentration are attributed to increasing lipid supersaturation under these operating conditions. Relative to formation of LNPs using ethanol (see, Example 2), the use of tethrahydrofuran for formation of LNPs permited further reduction in particle size, especially when the organic concentration after mixing was below 25% v/v.
  • LNPs are prepared by two variations of a two-stage particle formation and dilution process, while in one embodiment a one-stage process is used.
  • LNPs were prepared as follows. siRNA to luciferase was dissolved in citrate buffer (25 mM, 100 mM NaCl, pH 3.8) at 47 ⁇ M. Lipids (CLinDMA, PEG-DMG) and cholesterol were solubilized in tetrahydrofuran (THF) at a relative molar ratio of 60:38:2 (CLinDMA:Cholesterol:PEG-DMG) and a total lipid concentration of 6.7 mg/mL. To generate LNPs, the organic lipid solution was mixed with buffer solutions in a four-stream MIVM.
  • LNPs were prepared as follows. siRNA to luciferase was dissolved in citrate buffer (25 mM, 100 mM NaCl, pH 3.8) at 47 ⁇ M. Lipids (CLinDMA, PEG-DMG) and cholesterol were solubilized in tetrahydrofuran (THF) at a relative molar ratio of 60:38:2 (CLinDMA:Cholesterol:PEG-DMG) and a total lipid concentration of 6.7 mg/mL. To generate LNPs, the organic lipid solution was mixed with buffer solution in a first two-stream MIVM.
  • the flow rates of organic lipid solution and siRNA-containing buffer solution were each set at 22 mL/min. Mixing under these conditions yielded a particle suspension wherein the THF concentration after mixing was 50% v/v.
  • the particle suspension was immediately diluted by mixing with a controlled flow rate of buffer in a second MIVM mixing chamber.
  • particle suspension exiting the first MIVM was mixed against citrate buffer streams (25 mM, 100 mM NaCl, pH 3.8) using a four-stream MIVM.
  • the flow rates of the particle suspension was 44 mL/min, while the total flow rate of the citrate buffer diluent was 120 mL/min.
  • LNPs were prepared as follows. siRNA to luciferase was dissolved in citrate buffer (25 mM, 100 mM NaCl, pH 3.8) at 47 ⁇ M. Lipids (CLinDMA, PEG-DMG) and cholesterol were solubilized in tetrahydrofuran (THF) at a relative molar ratio of 60:38:2 (CLinDMA:Cholesterol:PEG-DMG) and a total lipid concentration of 6.7 mg/mL. To generate LNPs, the organic lipid solution was mixed with buffer solution in a two-stream MIVM.
  • the flow rates of organic lipid solution and siRNA-containing buffer solution were each set at 22 mL/min. Mixing under these conditions yielded a particle suspension wherein the THF concentration after mixing was 50% v/v.
  • the particle suspension was diluted by immediate introduction of the particle suspension into a stirred reservoir containing a controlled amount of buffer solution.
  • the reservoir contained an amount of buffer solution substantially greater than the amount of particle solution introduced thereto, such that the THF concentration after dilution was 13.4% v/v.
  • Aliquots of LNP suspension were dialyzed against phosphate buffered saline (PBS, pH 7) using a 6K-8K MWCO Spectra/Pore dialysis membrane. Dialysis was performed to exhange citrate buffer and to remove ethanol. Free (unencapsulated) siRNA does not pass through the dialysis membrane. Following dialysis, LNPs were characterized for particle size and siRNA encapsulation efficiency.
  • This Example serves to compare properties of LNPs prepared according to each of the three processes described.
  • the organic (e.g., THF) concentration in the particle suspension after mixing and dilution was 13.4% v/v.
  • the processes are distinguished primarily by the method by which the particle suspension after initial mixing was diluted.
  • dilution occurs coincidentally with particle formation.
  • particle formation occurs in the first mixing step and is followed in a second step by dilution.
  • dilution was found to be of critical importance, as LNP suspensions in solutions of high organic content (e.g. 50% v/v THF) were unstable when dilution was not effected. Particles quickly (e.g. within one minute) grew to macroscopic sizes (see, FIGS.9D , Sample 9.1 and 9E).
  • LNPs prepared by a one-stage mixing and dilution process (see, FIG. 9A ) in a four-stream MIVM were of small size (e.g. 54 nm) and possesed a narrow particle size distribution (see, FIGS. 9D , Sample 9.2 and 9E).
  • FIGS. 9D , Sample 9.2 and 9E A similar particle size was obtained when LNPs were prepared by a two-stage process in which particle formation was effected in a first MIVM mixing chamber and was followed by immediate dilution using a second MIVM mixing chamber (see, FIG. 9B ).
  • LNPs with a mean diameter of 57 nm were formed (see, FIG.
  • LNPs were prepared in accordance with a two-stage process in which particle formation occurred upon mixing in a first MIVM mixing chamber and was followed by dilution in a buffer resevoir (see, FIG. 9C ).
  • particles of large diameter e.g. 160 nm; see, FIG. 9D , Sample 9.4
  • very broad particle size distributions see, FIG. 9E .

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